The effect of purine stress on translation and protein stability in Leishmania

描述(申请人提供)：利什曼原虫是一种原生动物寄生虫，在人类中引起一系列疾病，是严重的人类健康负担。目前的治疗模式完全基于少数几种药物，这些药物可能有毒、昂贵和难以管理，其有效性因出现耐药性而受到破坏。由于成功的寄生依赖于生存的能力，即使环境条件，包括养分的可获得性，是不利的，利什曼原虫的营养胁迫反应途径为化疗探索提供了一个有吸引力的目标。然而，对促进这些寄生虫适应营养胁迫的信号通路或分子机制知之甚少。这项研究计划的长期目标是阐明利什曼原虫的营养应激反应途径。为了探索营养应激反应，我们使用了嘌呤饥饿作为一个范例。在利什曼狂症中，嘌呤的获得是一个基本的过程，在培养中很容易诱导嘌呤的饥饿，并引发既强健又容易驯服的反应。我们以前的研究表明，嘌呤的缺乏会导致寄生虫蛋白质组和代谢组的全面重组，从而导致深刻的形态和代谢变化。为了了解嘌呤饥饿后蛋白质组转化的分子机制，我们用RNA-seq的方法研究了嘌呤饥饿利什曼原虫的整体转录组的变化。这些研究表明，在嘌呤饥饿细胞的蛋白质组中，在mRNA水平上观察到的变化与在蛋白质组中表现出的变化之间存在显著的不一致，这意味着翻译和翻译后机制在嘌呤应激过程中的蛋白质组重塑中占主导地位。这一发展和探索性应用的目标是确定翻译的相对贡献 以及嘌呤饥饿过程中蛋白质组动力学的翻译后机制。我们将使用的方法涉及采用两种创新的代谢标记和蛋白质组学技术用于利什曼原虫，并将提供对嘌呤饥饿和嘌呤充足的利什曼原虫全球蛋白质合成的直接测量(目标1)，以及在蛋白质组范围内对蛋白质半衰期进行深入研究(目标2)。这是首次将这些技术用于利什曼原虫或其他相关病原体，这些研究解决了该领域的一个紧迫性，即提供对翻译和翻译后稳定性的直接和定量评估，这是基因调控的两个关键领域，已经在全球范围内进行了研究。 在这些寄生虫中。这些方法的成功实施也为这些寄生虫和其他寄生虫的未来研究奠定了基础，这些寄生虫有望比传统上提供的对翻译调控和蛋白质稳定性的更广泛的询问。最后，通过将这些系统水平的结果与我们之前的蛋白质组和RNA-SEQ数据相结合，我们将提供关于利什曼原虫和相关病原体适应微环境压力的分子机制的最全面的观点之一。

DESCRIPTION (provided by applicant): Leishmania are protozoan parasites that cause a spectrum of diseases in humans and are a significant human health burden. Current treatment paradigms are based exclusively on a handful of drugs that can be toxic, costly, and difficult to administer, and whose usefulness is undermined by the emergence of drug resistance. Since successful parasitism is contingent upon an ability to subsist even when environmental conditions, including nutrient availability, are unfavorable, nutrient-stress response pathways in Leishmania offer an attractive target for chemotherapeutic exploration. However, little is known about the signaling pathways or molecular mechanisms that facilitate adaptation to nutrient-stress in these parasites. The long-term goal of this research program is to elucidate nutrient stress response pathways in Leishmania. To probe nutrient stress response we have used purine starvation as a paradigm. Purine acquisition in Leishmania is an essential process and starvation for purines is readily induced in culture and provokes a response that is both robust and tractable. Our previous studies indicate that purine scarcity leads to profound morphological and metabolic changes that stem from an overall restructuring of the parasite proteome and metabolome. To comprehend the molecular mechanisms underlying proteome transformation in response to purine starvation, we have profiled changes in the global transcriptome of purine-starved Leishmania by the method of RNA-seq. These studies indicated that a significant discordance exists between changes observed at the mRNA level and those manifested within the proteome of purine-starved cells, implying that translational and post-translational mechanisms are predominant in proteome remodeling during purine stress. The goal of this developmental and exploratory application is to establish the relative contributions of translation and post-translational mechanisms on proteome dynamics during purine starvation. The approach that we will use involves adapting two innovative metabolic labeling and proteomic technologies for use in Leishmania, and will provide a direct measure of global protein synthesis in purine-starved and purine-replete Leishmania (Aim 1), as well as affording an in-depth study of protein half-life on a proteome-wide scale (Aim 2). This is the first documented use of these techniques in Leishmania or other related pathogens, and the studies address an exigency within the field, to provide both a direct and quantitative assessment of translation and post-translational stability, two key areas of gene regulation that have defied study on a global scale in these parasites. The successful implementation of these approaches also lays the foundation for future studies in these and other parasites that promise a much broader interrogation of translational regulation and protein stability than has traditionally been afforded. Finally, by th integration of these systems-level results with our previous proteome and RNA-seq data, we will offer one of the most inclusive views of the molecular mechanisms governing adaptation to micro-environmental stress in Leishmania and related pathogens.